Use of mixed mode chromatography for the capture and purification of basic antibody products

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Use of mixed mode chromatography for protein purification of an antibody from an antibody mixture, for example) a Pichia pastoris fermentation mixture containing impurities such as host cell proteins and DNA is described Mixed mode chromatography is used instead of protein A chromatography and presents certain advantages over protein A chromatography Furthermore, the integration of such a method into a multi-step procedure with other fractionation methods for purification of antibodies suitable for in vivo applications is provided.

Monoclonal antibodies are increasingly being developed and employed as therapeutics for a number of diseases, including autoimmune diseases, cancer, and infectious diseases, with over 200 antibodies in clinical trials. See Reichert, 2008, Curr Pharm Biotechnol 9:423-430.

However, monoclonal antibodies are among the most expensive of drugs with costs as high as $35,000 per year for a single patient. See Farid, 2007, J Chromatog B 848:8-18. The high cost associated with monoclonal antibodies is an impediment to its widespread usage. Therefore, reduction of costs associated with the clinical use of antibodies is critical.

One major contributor to these costs is the downstream processing, such as chromatography and filtration, necessary to purify the antibody from cell culture. See Kelley, 2007, Biotech Progress, 23: 995-1008. Downstream processing has been estimated to account for 50-80% of the total manufacturing costs of any antibody. See Roque, 2004, Biotechnol Prog 20:639-654. Due to their production in host cells, crude monoclonal antibody preparations contain many impurities, including cell components such as nucleic acids, proteins,polysaccharides, etc., as well as components of the culture media.

In a typical downstream processing scheme, a cell supernatant from cells used to produce a monoclonal antibody is clarified through the use of an initial protein purification step typically involving centrifugation or depth filtration. The clarified solution containing the monoclonal antibody is then separated from other proteins produced by the cell using a combination of different chromatography techniques, typically a capture chromatography step followed by

. i . polishing chromatography. See id. These techniques separate mixtures of proteins on the basis of their affinity for a biological or biomimetic ligand, charge, degree of hydrophobicity, or size. A final filtration step typically involving ultrafiltration or sterile filtration yields the final product. See id.

The capture chromatography step is commonly achieved by using affinity-based methods, which exploit a specific interaction between the antibody to be purified and an immobilized capture agent. Protein A chromatography, in particular, is typically used as the capture chromatography step in monoclonal antibody purifications. Protein A is a 41 kD ceil wall protein from Staphylococcus aureas which binds with a high affinity to the Fc region of antibodies. However, the use of protein A chromatography is associated with high cost.

Moreover, protein A generally cannot be cleaned with sodium hydroxide, adding to the cost associated with its use. Sodium hydroxide is a standard cleaning agent in preparative scale chromatography because it is inexpensive and effective at both cleaning and sanitizing the column for re-use. Furthermore, protein A chromatography typically requires elution at low pH, which can result in product aggregation and/or precipitation. When using protein A to purify antibodies made in yeast host cells, the protein A ligand itself is susceptible to yeast proteases, potentially resulting in higher levels of leached protein A and column deterioration.

New mixed mode chromatography ligands offer the potential for a next generation of protein purification processes by replacing a costly affinity capture step with a mixed-mode one. Mixed mode chromatography involves the use of solid phase chromatographic supports that employ multiple chemical mechanisms to adsorb proteins or other solutes. Examples of mixed mode chromatographic supports include but are not limited to chromatographic supports that exploit combinations of two or more of the following mechanisms: anion exchange, cation exchange, hydrophobic interaction, hydrophilic interaction, hydrogen bonding, pi-pi bonding, and metal affinity. Two such multi-modal ion exchange adsorbents are commercially available from GE Healthcare, the Capto Adhere™ and Capto MMC™ media. These combine strong anion and weak cation exchange groups, respectively, with hydrophobic aromatic groups.

Mixed mode chromatography supports provide unique selectivities that cannot be reproduced by single mode chromatography methods such as ion exchange. Mixed mode chromatography provides potential cost savings, longer column lifetimes and operation flexibility compared to affinity based methods. However, the development of mixed mode chromatography protocols can place a heavy burden on process development since multi-parameter screening is required to achieve their full potential. Method development is complicated, unpredictable, and may require extensive resources to achieve adequate recovery due to the complexity of the chromatographic mechanism.

What are needed are protein purification methods that provide for efficient purification and high yields of monoclonal antibodies.

Citation or identification of any reference in this section or any other section of this application shall not be construed as an indication that such reference is available as prior art to the present invention.